Modulation of the swelling-activated amino acid channel of Leishmania major promastigotes by protein kinases.
Leishmania promastigotes respond to hypotonic challenges by a mechanism of regulatory volume decrease (RVD), whereby anionic amino acid channels (HAAC) are hypotonically-activated and intracellular amino acids are released from the cells. Irrespective of the experimental conditions, restoration of isotonicity triggered an immediate blockage of the amino acid release. Both the speed and amplitude of the response depended on the hypotonic stimulus and on the operation of intracellular signaling mechanisms. The initial (5 s) hypotonic-induced release of amino acids (ri) and the steady state levels of amino acids attained (5 min) or amplitude (A), were markedly affected by modulators of protein kinase C: phorbol 12-myristate 13-acetate, 1-oleoyl-2-acetylglycerol and phorbol 12,13-diacetate whereas staurosporine and the related analog, bis-indolylmaleimide I (GF-109203.X) inhibited the RVD response. Agonists of cAMP-dependent protein kinase A such as forskolin or (8-(4-chlorophenylthio))-adenosine-3',5'cyclic-monophosphate enhanced the speed of the response but had little effect on its amplitude. Neither 4alpha-phorbol 12,13-didecanoate,1,9-dideoxyforskolin nor genistein, tamoxifen or thapsigargin had any apparent effect on either parameter tested. The most striking stimulation of hypotonic-induced amino acid release was exerted by arachidonic acid or by its non-metabolizable analog, 5,8,11,14-eicosatetraynoic acid (ETYA). These agents caused a major increase in the initial rate of amino acid release as well as a higher amplitude of the response, both of which were markedly inhibited by an anion channel blocker. The present studies indicate not only that hypotonicity is an obligatory and dominant component in HAAC activation, but implicate specific second messengers in the modulation of the RVD response. The modes of activation or attenuation of HAAC activity apparently differ for PKC and PKA modulators as well as for arachidonic acid. The involvement of Ca2+ in HAAC was studied in hypotonic challenged cells which were treated with intracellular Ca2+-chelators or Ca2+-free medium. These cells showed a lag in AA release and a modest inhibition of the amplitude. The inhibition of HAAC was markedly increased when cells were treated with the ionophore A23187 in Ca2+-free media. The HAAC activity was accompanied by a significant increase in internal Ca2+ when performed in Ca2+-containing medium (from 88+/-9 to 179+/-22 nM) but by no significant change when measured in Ca2+-free medium. These studies indicate that although Ca2+ might be involved in the early activation phase of HAAC, it is either not absolutely required or its action might be associated with localized events.
Vieira
L L
LL
Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem, Israel.
Lafuente
E
E
Blum
J
J
Cabantchik
Z I
ZI
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D018118:Chloride Channels / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005453:Fluorescence; D007038:Hypotonic Solutions; D007552:Isotonic Solutions; D018320:Leishmania major / Q000254:growth & development / Q000378:metabolism*; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D000596:Amino Acids; D018118:Chloride Channels; D007038:Hypotonic Solutions; D007552:Isotonic Solutions; D016718:Arachidonic Acid; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium